US5403731A - Process of producing modified superoxide dismutase - Google Patents

Process of producing modified superoxide dismutase Download PDF

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US5403731A
US5403731A US08/162,382 US16238293A US5403731A US 5403731 A US5403731 A US 5403731A US 16238293 A US16238293 A US 16238293A US 5403731 A US5403731 A US 5403731A
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sod
peg
reaction
ppg
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Yoshiyuki Nakano
Hajime Hiratani
Kazuo Kato
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JCR Pharmaceuticals Co Ltd
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JCR Pharmaceuticals Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/38—Drugs for disorders of the endocrine system of the suprarenal hormones
    • A61P5/40—Mineralocorticosteroids, e.g. aldosterone; Drugs increasing or potentiating the activity of mineralocorticosteroids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/08—Vasodilators for multiple indications
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33396—Polymers modified by chemical after-treatment with organic compounds containing nitrogen having oxygen in addition to nitrogen

Definitions

  • This invention relates to modified superoxide dismutases that are utilizable as pharmaceuticals, such as treatment agents of ischemic diseases or radiation hazards, and antitumor agents; in cosmetics intended for use in the prevention of oxygen injuries occurring on the skin; and for other purposes.
  • SOD Superoxide dismutase
  • SOD exists in three different types; SOD containing copper and zinc, SOD having manganese and extracellular SOD being present outside cells, among which the type containing copper and zinc is widely known to the general public. Reports have been made that such different types of SOD are effective for rheumatism and arthritis deformans, and in expectation of preventing injuries by ischemia- recirculation, for example, clinical trials are currently under way but in most cases through administration of SOD in the unmodified state.
  • This invention is concerned with a process which can permit modified superoxide dismutases with a high degree of purity having a markedly elongated blood half-life and consequently finding a wide range of clinical application as a drug substance to be produced on a commercial scale in simplified manners and in increased yields.
  • a great variety of chemical modifications have been performed in order to provide SOD with a prolonged half-life in the blood.
  • modification of SOD was made with high-molecular-weight dextrin (W. F. Petrone et al., Proc. Natl. Acad. Sci. U.S.A., 77, 1159 (1980)), polyethylene glycols (The Japanese Unexamined Patent Publication Nos.
  • SOD derivatives have all been developed for the purpose of preventing oxidative tissue injuries in the living body through intravenous and intramuscular administration. Except as stated in the above report by Charles O. Beauchamp et al., all of such derivatives are the high-molecular-weight modified SODs that are produced by use of activated modifying agents having two functional groups with the result that two molecules of SOD are introduced, and consequently, they are provided with an extremley extended half-life in the blood; they present the disadvantage that they remain in the living body for a MUCH TOO long period of time, although they offer the advantage of having a longer blood half-life than unmodified SOD.
  • the present invention is concerned with a process of producing modified superoxide dismutases represented by the formula: ##STR3## (wherein R is as defined below; SOD is a residue of superoxide dismutase), characterized in that said process comprises reacting a polymeric carbonyldiimidazole derivative represented by the formula: ##STR4## (wherein R is a residue of a water-soluble polymer having an average molecular weight of 2,000 to 10,000) with superoxide dismutase at a temperature of 30° to 70° C., preferably 45° to 60° C., in the presence of a buffer having a pH of 9.0 to 11.0 and a concentration of 0.1M to 0.5, preferably 0.2 to 0.4 M.
  • FIG. 1 is a graph showing the time-course changes of the blood levels of modified and unmodified superoxide dismutases in the animal experiment as described in Experiment Example 1.
  • FIG. 2 is a spectrophotogram of the modified superoxide dismutase as purified through gel permeation in Example 1.
  • the polymeric carbonylinidazole of the formula (I) can be obtained by reacting a water-soluble polymer having a molecular weight of about 2,000 to 10,000 with carbonyldiimdiazole (CDI) in an inert solvent such as dioxane.
  • CDI carbonyldiimdiazole
  • water-soluble polymer there may be mentioned, for example, polyoxyalkylene glycols having a molecular weight of about 2,000 to 10,000, such as polyoxyethylene glycols, mono-lower-alkoxypolyoxyethylene glycols and mono-lower-alkoxypolyoxyethylene.polyoxypropylene.polyoxyethylene glycols.
  • the above-mentioned lower axhoxys usually are C 1 to C 4 alkoxy groups.
  • Preferred examples of the water-soluble polymer include monomethoxypolyoxyethylene glycols having an average molecular weight of 3,500 and monomethoxypolyoxyethylene.polyoxypropylene.polyoxyethylene glycols having the same molecular weight.
  • the water-soluble polymer desirably is added into a reaction solvent in such a quantity as its initial concentration may range from 0.15 to 0.35M, preferably from 0.25 to 0.3M, while an initial concentration ratio of the polymer to CDI being at 1:1 to 3, preferably 1:1.5 to 2.5.
  • the ratio of activation of the polymer can be maintained at a fixed level through addition of a weakly acid buffer having a pH range of 6 to 6.5 to the reaction mixture to thereby allow the reaction to discontinue without increasing its pH.
  • the derivative the formula (I) can be separated from the reaction mixture by use of such separatory means as dialysis and lyophilization.
  • Table 1 shows the comparison of an example with this invention and the method of Beauchamp et al.
  • the decided differences between the process of the present inventors and the one of Beacuhamp et al. lie in the concentration of the modifying agent as well as the discontinuation of the reaction through addition of a buffer without bring about an increase in pH value, as employed and effected individually in the production of the activated modifying agent being shown in (A) of Table 1.
  • the improvement of the said two conditions are essential for the stable large-scale production of the activated modifying agent; namely, the optimally increased concentration of the modifying agent facilitates the activation in large quantities.
  • the range of the concentration can be selected from 0.15 to 0.35M, preferably from 0.25 to 0.3M.
  • Beuchamp et al. conducted investigation at 1:10 of the concentration ratio of the modifying agent to the activating agent CDI, the present inventors found out that when the said concentration ratio is maintained at 1:1 to 1:3, preferably 1:1.5 to 1:2.5, the modification can best be performed constant. Also, the increased concentration of the modifying agent can permit the volume of reaction solution to be reduced. This enables the utilization of the equipment capable of concentrating furthermore the reaction solution after conclusion of the reaction, resulting in simplified subsequent treatment steps.
  • Table 1 (B) in which the modification of SOD with activated PEG or PEG-PPG is described, can elucidate more clearly the characteristic features of this invention.
  • the increased concentration of the buffer makes it possible to minimize a change in pH due to a varying amount of the protein and furthermore that a rise in pH can accelerate the reaction rate.
  • the buffer desirably shows a pH of 9 to 10 and a concentration of 0.1 to 0.5M, preferably 0.2 to 0.4M.
  • the composition of the buffer is not particularly limited,, only if the buffer can have the buffering capacity over, the pH range of this invention and can be easily prepared.
  • Sodium carbonate buffer is preferable, because it is often used as an additive for pharmaceutical preparations and is considered highly safe.
  • the present inventors carried out extensive investigation into a large-scale production process in which the modification of conclusion of the reaction, resulting in simplified subsequent treatment steps.
  • Table 1 (B), in which the modification of SOD with activated PEG or PEG-PPG is described, can elucidate more clearly the characteristic features of this invention.
  • the buffer desirably shows a pH of 9 to 10 and a concentration of 0.1 to 0.5M, preferably 0.3 to 0.4M.
  • the composition of the buffer is not particularly limited, only if the buffer can have the buffering capacity over the pH range of this invention and can be easily prepared.
  • Sodium carbonate buffer is preferable, because it is often used as an additive for pharmaceutical preparations and is considered highly safe.
  • the present inventors carried out extensive investigation into a large-scale production process in which the modification reaction of SOD with activated PEG or PEG-PPG can be proceeded faster, and as a result, found that the reaction at temperatures of 30° to 70° C., preferably 45° to 60° C., can lead to completion of the modification within a extremely shortened period of time.
  • the modification reaction can be carried out with the concentrations of both activated PEG or PEG-PPG and SOD being increased, thus making the commercial, large-scale production practically feasible.
  • anion exchanger column chromatography is a means being effective for the entire elimination of contamination with unreacted activating agent. Since the unmodified activating agent and the modified SOD have individually different molecular weights of 32,000 and 130,000, it usually is a common practice to employ molecular sieve column chromatography, but the present inventors found that anion exchanger column chromatography, being more efficient, is best suited for the said purpose.
  • any anion exchangers being utilizable to this effect, use can be made of any anion exchangers, only if they possess DEAE groups, but preferably DEAE-Sepharose CL-6B may be usable. It was found out that the SOD being modified in this manner with activated PEG or PEG-PPG shows a ratio of modification as constant as 17 to 20% and that such modified SOD can be produced more efficiently than in the case of the method of Beauchamp et al. when, a chemical compound is intended for use as a pharmaceutical, considered to be the most desirable is the production process being capable of securing the constant, and in the light of the characteristics as described above, the process according to this invention can be said to fully meat such requirement.
  • SOD that is usable in this invention is not particularly limited in terms of its origin or source, but is desirably SOD containing copper and zinc.
  • the retention (%) of the enzymatic activity of modified SOD was expressed in terms of a proportion on the basis of the enzymatic activity (taken as 100%) of unmodified SOD as assayed against the superoxide generated in the xanthine-xanthine oxidase system.
  • the protein was reacted with divalent copper under alkaline conditions, and the resulting red-purple reaction solution was subjected to measurement of absorbance at a wavelength of 540 nm.
  • the protein was determined quantitative through calculation from a calibration curve prepared with bovine serum albumin used as a standard.
  • House rabbits being used as experimental animals, were given intravenously SOD modified with PEG-PPG (a molecular weight of 3,500) and unmodified SOD, as replaced with or dissolved in isotonic saline, at a dose of 38,000 units per 2.5 kg body weight, respectively, followed by time-course determination of the serum SOD levels.
  • the results are shown in FIG. 1, which indicates that the modified SOD, showing a lowered rate of blood clearance as compared with unmodified SOD, extended a length of the time of SOD activity development in the blood.
  • Unmodified SOD or modified SOD (ca. 30 ug each) was emulsified with Freund Complete Adjuvant (FCA) and was given A/J mice intraperitoneally, followed by additional administration on Day 14 and Day 28 for immunization.
  • FCA Freund Complete Adjuvant
  • reaction solution containing a CDI derviative of OMe-PEG-PPG (hereinafter referred to as "CDI-PEG-PPG").
  • CDI-PEG-PPG a reaction solution containing a CDI derviative of OMe-PEG-PPG
  • the reaction solution was concentreated under reduced pressure over a water bath at a temperature of lower than 30° C. to give about 110 ml of a highly viscous concentrate, to which 0.5M sodium phosphate buffer (pH 6.5) was then added to make up to 200 ml.
  • the diluted solution was subjected to dialysis treatment (the outer solution: water) to give 1,000 ml of the sample solution, followed by lyophilization to produce CDI-PEG-PPG in the form of dried powder, which was stored at -30° C. Yield of 98%.
  • the adsorbed PEG-PPG-SOD was eluted with 0.3M sodium carbonate-hydrochloric acid buffer (pH 9.5), and the eluate was concentrated through ultrafiltration.
  • the concentrate obtained through ultrafiltration under the section (3) was adjusted to a concentration of 50 ⁇ 5 mg/ml and subjected to the same procedure as performed under the section (2) to give a crude solution of SOD completely modified with PEG-PPG, which was then chromatographed on a column of DEAE-Sephrose by the same procedure as described under the section (3).
  • the elution was performed with 25 mM sodium phosphate buffer (pH 7.0) containing 0.9% sodium chloride, and the eluate was concentrated through ultrafiltration and sterile-filtered to give a pure PEG-PPG-SOD solution. Yield of 4.8 g or 96%.
  • the product was found to be a pure and single compound as evidenced by TSK G 3000SW gel permeation and electrophoresis analyses.
  • reaction solution containing a CDI derivative of PEG-PPG (CDI-PEG-PPG).
  • reaction solution was concentrated under reduced pressure over a water bath at a temperature of lower than 30° C. to produce about 600 ml of a highly viscous concentrate, followed by addition of 0.5M sodium phosphate buffer (pH 6.5) to dilute to 1000 ml.
  • the diluted solution was subjected to dialysis treatment (the outer solution: water) to give 5000 ml of a sample solution, followed by lyophylization to produce CDI-PEG-PPG in the form of dried powder.
  • the product was stored at -30° C. Yield of 99%.
  • the crude PEG-PPG-SOD solution as obtained under the section (2) was poured for adsorption into a column packed with DEAE-Sepharose CL-6B (produced by Pharmacia Co.) which had been equilibrated through thorough washing with sufficient volume of water, and the column was washed with water offive times the volume of the column to remove unreacted CDI-PEG-PPG.
  • the adsorbed PEG-PPG-SOD was eluted with 0.3M sodium carbonate buffer (pH 9.5), and the eluate was concentrated through ultrafiltration.
  • the concentrate obtained; through ultrafiltration under the section (3) was adjusted to a concentration of 50 ⁇ 5 mg/ml and subjected to the same procedure as described in the section (2) to give a crude solution of SOD completely modified with PEG-PPG.
  • the crude PEG-PPG-SOD solution was chromatographed on a column of DEAE-Sepharose in the same manner as described under the section (3), followed by elution with 2.5 mM sodium phosphate buffer (pH 7.0) containing 0.9% of sodium chloride, and the eluate was comcentrated through ultrafiltration and sterile filtered to give a pure PEG-PPG-SOD solution. Yield of 29 g or 97%. The product was found to be pure and single compound as evidence by electrophoresis analysis.
  • the crude PEG-SOD Solution as obtained under section (2) was poured for adsorption into a column packed with DEAE-Toyo Pearl (produced by Toso Inc. of Japan) which had been equilibrated through thorough washing with sufficient volume of water, and the column was washed with water of five times the volume of the column ro remove unreacted CDIPEG.
  • the adsorbed PEG-SOD was eluted with 0.3M sodium carbonate buffer (pH 9.5), and the eluate was concentrated through ultrafiltration.
  • the concentrate obtained through ultrafiltration under the section (3) was adjusted to a concentration of 50 ⁇ 5 mg/ml and subjected to same procedure as described under the section (2) to give a crude solution of SOD completely modified with PEG.
  • the crude PEG-SOD solution was further chromatographed on a column of DEAE-Toyo Pearl in the same manner as described in the section (3), followed by elution with 25 mM sodium phosphate buffer (pE 7.0) containing 0.9% of sodium chloride., and the eluate was concentrated through ultrafiltration and sterile filtered to give a pure PEG-SOD solution. Yield of 4.9 g or 98%.
  • the product was found to be a pure and single compound as evidenced by gel permeation on TSKG 3000 SW and electrophoresis.
  • the diluted solution was subjected to dialysis treatment (the outer solution: water) to give 1900 ml of a sample solution, followed by lyophilization to produce CDI-PEG-PPG in the form of dried powder.
  • the product was stored at -30° C. Yield of 94%.
  • the crude PEG-PPG-SOD solution as obtained under the section (2) was poured for adsorption into a column packed with DEAE-Sepharose CL-6B (produced by Pharmacia. Co.) which had been equilibrated through thorough washing with sufficient volume of water, and the column was washed with water of five times the volume of the column to remove unreacted CDI-PEG-PPG.
  • the adsorbed PEG-PPG-SOD was eluted with 0.3M sodium carbonate buffer (pH 9.5), and the eluate was concentrated through ultrafiltration to give about 220 ml of a concentrate, followed by sterile filtration to give 9.85 g of modified SOD solution. Yield of 98,.5%. Gel permeation on TSKG 3000 SW and electrophoresis showed that unreacted SOD was not detected at all.
  • the diluted solution was subjected to dialysis treatment (the other solution: water) to give 1000 ml of a sample solution, followed by lyophilization to produce CDI-PEG-PPG in the form of dried powder.
  • the product was stored at -30° C.
  • the crude PEG-PPG-SOD solution as obtained under the section (2) was poured for adsorption into a column packed with DEAE-Sepharose CL-6B (produced by Pharmacia Co.) which had been equilibrated through thorough washing with sufficient volume of water, and the,column was washed with water of five times the volume of column to remove unreacted CDI-PEG-PPG.
  • the adsorbed PEG-PPG-SOD was eluted with 0.3M sodium carbonate buffer (pH 9.5), and the eluate was concentrated through ultrafiltration to give 115 ml of a concentrate, followed by sterile filtration to give 4.89 g of a modified SOD solution. Yield of 97.8%. Gel permeation on TSK G 3000 SW and electrophoresis showed that unmodified SOD was not detected at all.

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US08/162,382 1989-11-02 1993-12-03 Process of producing modified superoxide dismutase Expired - Fee Related US5403731A (en)

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JP1286798A JP2978187B2 (ja) 1989-11-02 1989-11-02 修飾スーパーオキサイドディスムターゼの製造法
US60819690A 1990-11-02 1990-11-02
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US6190658B1 (en) 1998-05-08 2001-02-20 Webb-Waring Institute For Biomedical Research Genetically modified manganese superoxide dismutase for treating oxidative damage
US20030027745A1 (en) * 2001-06-13 2003-02-06 Repine John E. Diagnostic and prognostic method for evaluating ocular inflammation and oxidative stress and the treatment of the same
US20100187700A1 (en) * 2006-08-31 2010-07-29 Karl Weidner Method and apparatus for manufacturing an electronic module, and electronic module
CN109112119A (zh) * 2018-08-28 2019-01-01 佛山科学技术学院 经化学修饰的鸭血sod制剂的制备方法

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JP3125428B2 (ja) * 1991-03-28 2001-01-15 和光純薬工業株式会社 修飾酵素
US5382657A (en) * 1992-08-26 1995-01-17 Hoffmann-La Roche Inc. Peg-interferon conjugates
US5650234A (en) * 1994-09-09 1997-07-22 Surface Engineering Technologies, Division Of Innerdyne, Inc. Electrophilic polyethylene oxides for the modification of polysaccharides, polypeptides (proteins) and surfaces
US5824784A (en) 1994-10-12 1998-10-20 Amgen Inc. N-terminally chemically modified protein compositions and methods
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US7192560B2 (en) 2001-12-20 2007-03-20 3M Innovative Properties Company Methods and devices for removal of organic molecules from biological mixtures using anion exchange
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US5006333A (en) * 1987-08-03 1991-04-09 Ddi Pharmaceuticals, Inc. Conjugates of superoxide dismutase coupled to high molecular weight polyalkylene glycols

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Cited By (5)

* Cited by examiner, † Cited by third party
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US6190658B1 (en) 1998-05-08 2001-02-20 Webb-Waring Institute For Biomedical Research Genetically modified manganese superoxide dismutase for treating oxidative damage
US20030027745A1 (en) * 2001-06-13 2003-02-06 Repine John E. Diagnostic and prognostic method for evaluating ocular inflammation and oxidative stress and the treatment of the same
US20100187700A1 (en) * 2006-08-31 2010-07-29 Karl Weidner Method and apparatus for manufacturing an electronic module, and electronic module
CN109112119A (zh) * 2018-08-28 2019-01-01 佛山科学技术学院 经化学修饰的鸭血sod制剂的制备方法
CN109112119B (zh) * 2018-08-28 2022-04-26 佛山科学技术学院 经化学修饰的鸭血sod制剂的制备方法

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JPH03147784A (ja) 1991-06-24
EP0426488B1 (de) 1997-04-23
DE69030553T2 (de) 1998-01-22
ES2100165T3 (es) 1997-06-16
DE69030553D1 (de) 1997-05-28
JP2978187B2 (ja) 1999-11-15
CA2029216A1 (en) 1991-05-03

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